Linking sequence reads using paired code tags

ABSTRACT

Artificial transposon sequences having code tags and target nucleic acids containing such sequences. Methods for making artificial transposons and for using their properties to analyze target nucleic acids.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledILLINC193ASEQLIST.TXT, created Feb. 10, 2011, which is approximately 2Kb in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Embodiments of the present invention relate to the fields of biology andgenomics. Some embodiments of the present invention relate to methodsand compositions that include certain transposon sequences. Some suchmethods and compositions include analyzing target nucleic acids.

BACKGROUND OF THE INVENTION

The detection of specific nucleic acid sequences present in a biologicalsample has been used, for example, as a method for identifying andclassifying microorganisms, diagnosing infectious diseases, detectingand characterizing genetic abnormalities, identifying genetic changesassociated with cancer, studying genetic susceptibility to disease, andmeasuring response to various types of treatment. A common technique fordetecting specific nucleic acid sequences in a biological sample isnucleic acid sequencing.

Nucleic acid sequencing methodology has evolved significantly from thechemical degradation methods used by Maxam and Gilbert and the strandelongation methods used by Sanger. Today several sequencingmethodologies are in use which allow for the parallel processing ofnucleic acids all in a single sequencing run. As such, the informationgenerated from a single sequencing run can be enormous.

SUMMARY OF THE INVENTION

Some embodiments of the present invention relate to methods andcompositions that include certain transposon sequences. Some suchmethods and compositions include analyzing target nucleic acids. Someembodiments include methods of preparing a template nucleic acid. Somesuch methods include: (a) providing a target nucleic acid; (b) providinga plurality of transposon sequences, each transposon sequence comprisinga first transposase recognition site, a second transposase recognitionsite having a barcode disposed therebetween; and (c) contacting thetarget nucleic acid with the plurality of transposon sequences underconditions such that at least a portion of said plurality of transposonsequences inserts into the target nucleic acid, thereby preparing atemplate nucleic acid.

In some embodiments, the barcode of each transposon is different.

In some embodiments, the barcode comprises a first barcode sequence, asecond barcode sequence, said first and second barcode sequences beingseparated by a linker disposed therebetween.

In some embodiments, the linker comprises a nucleic acid.

In some embodiments, the linker comprises a sequencing adaptercomprising a first primer site.

In some embodiments, the linker comprises a fragmentation site. In someembodiments, the fragmentation site comprises a first nickaserecognition sequence and a second nickase recognition sequence, whereinthe cut site for each recognition sequence is the same site. In someembodiments, the fragmentation site comprises a restriction endonucleaserecognition sequence.

In some embodiments, the linker comprises a sequencing adaptercomprising a first primer site and a fragmentation site. In someembodiments, the linker comprises a sequencing adapter comprising afirst primer site and a second primer site having a fragmentation sitedisposed therebetween.

Some embodiments include methods of preparing a library of templatenucleic acids. Some such methods include: (a) providing a target nucleicacid; (b) providing a plurality of transposon sequences, each transposonsequence comprising a first transposase recognition site and a secondtransposase recognition site having a barcode disposed therebetween,wherein the barcode comprises a first barcode sequence and a secondbarcode sequence being separated by a sequencing adapter disposedtherebetween, wherein the sequencing adapter comprises a first primersite and a second primer site; and (c) contacting the target nucleicacid with the plurality of transposon sequences under conditions suchthat at least a portion of said plurality of transposon sequencesinserts into the target nucleic acid; and (d) amplifying at least aportion of the target nucleic acid by hybridizing a primer to said firstprimer site and a primer to said second primer site, such that theamplification product comprises a first barcode sequence and a secondbarcode sequence, thereby preparing a library of template nucleic acids.

In some embodiments, the barcode of each transposon is different.

Some methods also include a step subsequent to (c) and prior to (d)comprising reducing the number of target nucleic acid moleculescomprising inserted transposon sequences.

In some embodiments, the sequencing adapter comprises a first primersite, a second primer site, and a non-amplifiable site therebetween. Insome embodiments, the non-amplifiable site comprises a nucleic acid. Insome embodiments, the non-amplifiable site comprises at least onenucleotide analogue. In some embodiments, the nucleotide analogue doesnot significantly basepair with A, C, G or T.

Some embodiments include methods of preparing a library of templatenucleic acids. Some such methods include: (a) providing a target nucleicacid; (b) providing a plurality of transposon sequences, each transposonsequence comprising a first transposase recognition site and a secondtransposase recognition site having a barcode disposed therebetween,wherein the barcode comprises a first barcode sequence and a secondbarcode sequence being separated by a linker disposed therebetween,wherein the linker comprises a fragmentation site; (c) contacting thetarget nucleic acid with the plurality of transposon sequences underconditions such that at least a portion of said plurality of transposonsequences inserts into the target nucleic acid; and (d) fragmenting saidtarget nucleic acid at said fragmentation sites, such that at least aportion of the fragmented nucleic acids each comprise a first barcodesequence and a second barcode sequence, thereby preparing a library oftemplate nucleic acids.

In some embodiments, the barcode of each transposon is different.

In some embodiments, the linker comprises a nucleic acid.

In some embodiments, the fragmentation site comprises a first nickaserecognition sequence, a second nickase recognition sequence, wherein thecut site for each recognition sequence is the same site.

In some embodiments, the fragmentation site comprises a restrictionendonuclease recognition sequence.

Some methods also include ligating a first primer site to a first end ofat least one fragmented nucleic acid. Some methods also include ligatinga second primer site to the second end of the at least one fragmentednucleic acid.

Some methods also include amplifying said at least one fragmentednucleic acid by hybridizing a primer to the first primer site and aprimer to the second primer site.

In some embodiments, the linker further comprises a first primer site.

In some embodiments, the linker comprises a sequencing adapter comprisesa first primer site, a second primer site having the fragmentation sitedisposed therebetween. Some methods also include amplifying said atleast a portion of the fragmented nucleic acids by hybridizing a primerto said first primer site and a primer to said second primer site.

In some embodiments, the target nucleic acid comprises genomic DNA.

In some embodiments, the first transposase recognition site comprises amosaic element.

In some embodiments, the sequences of the first barcode and the secondbarcode comprise the reverse complements of each other.

Some embodiments include libraries of template nucleic acids prepared byany one of the foregoing methods.

Some embodiments include methods of preparing a transposon sequence.Some such methods include: (a) providing a transposon template nucleicacid sequence comprising a first transposase recognition site, a firstbarcode 3′ thereof, and a linker 3′ thereof; and (b) extending thetransposon template sequence by hair-pin extension, such that theextended transposon template sequence further comprises sequencescomplementary to said barcode and to said transposase recognition site,thereby preparing a transposon comprising a first transposaserecognition site, a first barcode, a linker, a second barcode, and asecond transposase recognition site.

In some embodiments, the barcode comprises a random sequence.

In some embodiments, the transposase recognition site comprises a mosaicelement.

In some embodiments, the barcode comprises at least about fivenucleotides.

In some embodiments, the linker comprises a nucleic acid.

In some embodiments, the linker comprises a fragmentation site. In someembodiments, the fragmentation site comprises a first nickaserecognition sequence, a second nickase recognition sequence, wherein thecut site for each recognition sequence is the same site. In someembodiments, the fragmentation site comprises a restriction endonucleaserecognition sequence.

In some embodiments, the linker comprises a sequencing adapter. In someembodiments, a sequencing adapter comprises a first primer site andsecond primer site. In some embodiments, the sequencing adaptercomprises a first primer site and a second primer site having afragmentation site disposed therebetween. In some embodiments, thesequencing adapter comprises a first primer site and a second primersite, having a non-amplifiable site disposed therebetween.

In some embodiments, the non-amplifiable site comprises a nucleic acid.In some embodiments, the non-amplifiable site comprises at least onenucleotide analogue. In some embodiments, the nucleotide analogue doesnot significantly basepair with A, C, G or T.

Some embodiments include methods of preparing a plurality of transposonsequences. Some such method include repeating steps (a) and (b) of anyone of the foregoing methods of preparing a transposon sequence, whereinthe barcode of each transposon template nucleic acid is different.

Some embodiments include transposons prepared by the method of any oneof the foregoing methods of preparing a transposon sequence.

Some embodiments include methods of analyzing a target nucleic acid.Some such methods include: (a) providing a template nucleic acid,wherein the template nucleic acid comprises the target nucleic acid anda plurality of markers inserted therein, wherein each marker sequencecomprises a barcode comprising a first barcode sequence and a secondbarcode sequence, having a sequencing adapter disposed therebetween,wherein the sequencing adapter comprises a first primer site; (b)obtaining sequence data from said template nucleic acid; and (c)assembling a representation of at least a portion of said target nucleicacid from said sequence data.

In some embodiments, the barcode of each marker is different.

In some embodiments, obtaining sequence data comprises hybridizing aprimer to said first primer site, and extending said primer.

In some embodiments, the sequencing adapter comprises a second primersite, and said obtaining sequence data comprises hybridizing a primer tothe second primer site and extending said primer. In some embodiments,the primers hybridize to the sites in opposite orientations.

In some embodiments, the sequences of the first barcode sequence and thesecond barcode sequence comprise the reverse complements of each other.

In some embodiments, the assembling step comprises identifying more thanone sequencing read comprising the first barcode sequence or secondbarcode sequence of a barcode.

In some embodiments, the presence of a first barcode sequence or secondbarcode sequence of a barcode in more than one sequencing read isindicative of the more than one sequencing reads representing sequencesadjacent to each other in the target nucleic acid.

In some embodiments, each marker sequence comprises a first host tag andsecond host tag having the barcode disposed therebetween. In someembodiments, the first and second host tag of a marker comprises thesame sequence. In some embodiments, the assembling step furthercomprises identifying more than one sequencing read comprising the samehost tags.

In some embodiments, the target nucleic acid comprises genomic DNA.

Other embodiments include artificial transposon sequences. Some suchartificial transposon sequences include: a first transposase recognitionsite and a second transposase recognition site having a barcode disposedtherebetween, wherein the barcode comprises a first barcode sequence anda second barcode sequence, said first and second barcode sequences beingseparated by a linker.

In some embodiments, the first transposon recognition site comprises amosaic element.

In some embodiments, the linker comprises a nucleic acid.

In some embodiments, the linker comprises a fragmentation site. In someembodiments, the fragmentation site comprises a first nickaserecognition sequence and a second nickase recognition sequence, whereinthe cut site for each recognition sequence is the same site. In someembodiments, the fragmentation site comprises a restriction endonucleaserecognition sequence.

In some embodiments, the linker comprises a sequencing adaptercomprising a first primer site. In some embodiments, the sequencingadapter comprises a second primer site. In some embodiments, thesequencing adapter comprises a first primer site and a second primersite, having a non-amplifiable site disposed therebetween. In someembodiments, the non-amplifiable site comprises a nucleic acid. In someembodiments, the non-amplifiable site comprises at least one nucleotideanalogue. In some embodiments, the nucleotide analogue does notsignificantly basepair with A, C, G or T.

In some embodiments, the linker comprises a sequencing adaptercomprising a first primer site and a second primer site, having afragmentation site disposed therebetween.

In some embodiments, a sequencing primer is hybridized to said firstprimer site.

In some embodiments, a sequencing primer is hybridized to said firsttransposase recognition site.

Some embodiments include populations of artificial transposon sequencescomprising a plurality of any one of the foregoing artificial transposonsequences.

Some embodiments include target nucleic acids or copies thereof having apopulation of the artificial transposon sequences integrated therein,wherein the artificial transposon sequences comprise any one of theartificial transposon sequences described herein.

Some embodiments include populations of genomic DNA fragments or copiesthereof having a population of the artificial transposon sequencesintegrated therein, wherein the artificial transposon sequences compriseany one of the artificial transposon sequences described herein.

Some embodiments include genomes having a population of artificialtransposon sequences integrated therein, wherein the artificialtransposon sequences comprise any one of the artificial transposonsequences described herein.

Some embodiments include isolated template nucleic acids. Some suchisolated template nucleic acids include at least a portion of a targetnucleic acid or copy thereof and at least two markers inserted therein,wherein each marker sequence comprises a barcode.

In some embodiments, the barcode of each marker is different.

Some embodiments also include a transposase recognition site.

Some embodiments also include a first transposase recognition site and asecond transposes recognition site, having the at least a portion of atarget nucleic acid or copy thereof disposed therebetween.

Some embodiments also include a host tag. Some embodiments also includea first host tag and a second host tag, having the at least a portion ofa target nucleic acid or copy thereof disposed therebetween. Someembodiments also include a first host tag and a second host tag, havinga transposase recognition site disposed therebetween, wherein thetransposase recognition site comprises a first transposase recognitionsite and a second transposase recognition site, having the at least aportion of a target nucleic acid or copy thereof disposed therebetween.

In some embodiments, the host tag comprises nine nucleotides.

In some embodiments, the target nucleic acid comprises genomic DNA.

Some embodiments include a plurality of template nucleic acidscomprising the template nucleic acid of any one of the isolated templatenucleic acids described herein, wherein a first template nucleic acid ofsaid plurality comprises a first barcode, and a second template nucleicacid comprises a second barcode, wherein the first and second barcodesare indicative of template nucleic acid sequences being adjacent to oneanother in a sequence representation of the target nucleic acid. In someembodiments, the first barcode comprises the reverse complement sequenceof the second barcode.

Some embodiments include substrates comprising nucleotide sequencesattached thereto, at least one of said nucleotide sequences comprisingany one of the template nucleic acids described herein, or any one ofthe plurality of template nucleic acids described herein. In someembodiments, the substrate can include a solid support selected from thegroup consisting of spheres, microparticles, beads, membranes, slides,plates, micromachined chips, tubes, microwells, microfluidic devices,channels, and filters.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic a transposon sequence. M indicates a firsttransposon recognition sequence. C1 indicates a first barcode sequence.The linker sequence is indicated by a rectangle with a double border.C1′ indicates a second barcode sequence. M′ indicates a secondtransposase recognition sequence. FIG. 1 also depicts exemplary linkersequences. For example, linker sequences can have fragmentation sites,such as those with nickase recognition sites (e.g. N1, N2) or arestriction endonuclease recognition sequence (indicated by RE). Inother embodiments, linkers can contain one or more primer sites (e.g. A,B), corresponding to complementary primers A′ and B′. A non-amplifiablesite is indicated by nA.

FIG. 2 depicts an exemplary scheme to prepare a transposon sequence.

FIG. 3 depicts the integration of an exemplary Tn5 transposon comprisingtransposase recognition sequences (indicated by mosaic elements ME) intoa target nucleic acid. The insertion results in duplication of theintegration site, shown as single-stranded A′B′C′D′E′F′G′H′I′ andsingle-stranded ABCDEFGHI. The figure also shows an optional repair stepto fill in the single-stranded region. In some embodiments, thefilled-in region can be used as a host tag.

FIG. 4 depicts an exemplary embodiment of a contacting a target nucleicacid with a library of transposons, which integrates into the targetnucleic acid.

FIG. 5 depicts an optional step of fragmenting a linker into two parts,or an optional step of virtually fragmenting the linker by amplificationusing a primer A′ and/or a primer B′. Fragmentation of the linkergenerates a library of template nucleic acids. In this figure, thefilled-in region is indicated by a thick line.

FIG. 6 depicts a schematic representation of using paired bar codes(indicated by Code 1, Code 2, Code 5) for code-pair sequencing oftemplate contig sequences to assemble the sequence of an original targetnucleic acid.

FIG. 7 depicts a graph showing that the proportion of template nucleicacids that are useful can decrease as the average distance between sitesof integration increases.

FIG. 8 depicts an exemplary embodiment of a method includingfragmentation.

FIG. 9 depicts an exemplary embodiment of a method that includesoptional steps of amplifying a target nucleic acid by PCR or wholegenome amplification (WGA). In this figure, the filled-in region isindicated by N₁ and N₂.

FIG. 10 depicts an exemplary embodiment of a method for assembling shortsequencing reads using a linked read strategy. Optional steps ofsub-sampling, code alignment, read assembly, and genome assembly arealso illustrated.

FIG. 11 depicts an embodiment using restriction endonucleases togenerate randomer sticky ends. Steps include: transposase insertstransposon into host material; dilution and whole genome amplification;fragmentation and linked code formation; restriction endonuclease nicksat indicated sites; and separation of randomer sticky ends.

FIG. 12 depicts a library preparation method that involves ligation of aforked adapter sequence. Steps include: library preparation; extensionand A-tailing; adapter ligation; and PCR amplify, cluster, and sequenceproducts.

DETAILED DESCRIPTION

Some embodiments of the present invention relate to methods andcompositions that include certain transposon sequences. Some suchmethods and compositions include analyzing target nucleic acids.Generally, methods of analyzing nucleic acids include preparing alibrary of template nucleic acids of a target nucleic acid, obtainingsequence data from the library of template nucleic acids, and assemblinga sequence representation of the target nucleic acid from such sequencedata. However, assembly of a sequence representation using traditionalmethods is met with several challenges. For example, sequencing data isobtained using short reads and thus there is difficulty assemblingcontiguous reads through repetitive sequences of a target nucleic acid.In addition, many contigs are required to be constructed to represent agenome such as the human genome. Methods such as paired-end sequencingcan be used to mitigate the difficulties of assembling sequence datafrom many short reads. However, the paired-end sequencing methodsrequire longer template nucleic acids to span repetitive sequences in atarget nucleic acid; preparation of template nucleic acids isinefficient and thus requires larger amounts of DNA; the number ofdifferent paired-ends is limited; and a reference genome is required toverify any resulting sequence representation.

Some nucleic acids of interest, such as genomic DNAs, comprise longmolecules with ordered sequence. Methods to sequence such molecules tendto be highly parallelized and include sequencing short libraries inorder to create high throughput. Consequently, such methods requireassembly of the shorter reads to obtain ordering information, i.e., asequence representation of the target nucleic acid. Shotgun sequencingapproaches uses DNA fragmentation; however, order information is lostduring fragmentation. Advantageously, some methods and compositionsprovided herein can be used to obtain shorter reads of template nucleicacids in which the order information is preserved. Thus, assembly ofshorter reads can be performed without the requirement of a referencegenome.

In an exemplary embodiment, a library of template nucleic acids isprepared from a target nucleic acid. The library is prepared byinserting a plurality of unique barcodes throughout the target nucleicacid.

As will be understood, although a barcode is frequently depicted in thefigures as a double-stranded, annealed structure, a useful barcode canbe a double-stranded region where the two strands are not complementaryor not annealed. For example, two noncomplementary regions can be linkedinformatically, even though they are not complementary in a conventionalWatson-Crick base-pairing.

Each barcode includes a first barcode sequence and a second barcodesequence, having a fragmentation site disposed therebetween. The firstbarcode sequence and second barcode sequences can be identified ordesignated to be paired with one another. The pairing can be informaticso that a first barcode is associated with a second barcode. The pairingcan also be physically associated to form a junction between the twobarcodes. For example, the first barcode sequence and second barcodesequences can be the tandem or reverse complements of each other. Thetarget nucleic acid can be fragmented at the fragmentation sites, and alibrary of template nucleic acids can be prepared from the fragments.Sequencing information can be obtained from the library of templatenucleic acids. Advantageously, the paired barcode sequences can be usedto assemble sequencing data from the library of template nucleic acids.For example, identifying a first template nucleic acid comprising afirst barcode sequence and a second template nucleic acid comprising asecond barcode sequence that is paired with the first indicates that thefirst and second template nucleic acids represent sequences adjacent toone another in a sequence representation of the target nucleic acid.Thus, a sequence representation of the target nucleic acid can beassembled by identifying further sequencing reads comprising pairedbarcode sequences. Such methods can be used to assemble a sequencerepresentation of a target nucleic acid de novo, without the requirementof a reference genome. Moreover, such methods are also useful tosequence target nucleic acids comprising highly repetitive sequences.The methods can also be used to reassemble alternative isoforms andsplice junctions in cDNAs, as well as reassemble single-moleculehaplotypes.

DEFINITIONS

As used herein the term “nucleic acid” and/or “oligonucleotide” and/orgrammatical equivalents thereof can refer to at least two nucleotidemonomers linked together. A nucleic acid can generally containphosphodiester bonds, however, in some embodiments, nucleic acid analogsmay have other types of backbones, comprising, for example,phosphoramide (Beaucage, et al., Tetrahedron, 49:1925 (1993); Letsinger,J. Org. Chem., 35:3800 (1970); Sprinzl, et al., Eur. J. Biochem., 81:579(1977); Letsinger, et al., Nucl. Acids Res., 14:3487 (1986); Sawai, etal., Chem. Lett., 805 (1984), Letsinger, et al., J. Am. Chem. Soc.,110:4470 (1988); and Pauwels, et al., Chemica Scripta, 26:141 (1986),incorporated by reference in their entireties), phosphorothioate (Mag,et al., Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No.5,644,048), phosphorodithioate (Briu, et al., J. Am. Chem. Soc.,111:2321 (1989), incorporated by reference in its entirety),O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press, incorporatedby reference in its entirety), and peptide nucleic acid backbones andlinkages (see Egholm, J. Am. Chem. Soc., 114:1895 (1992); Meier, et al.,Chem. Int. Ed. Engl., 31:1008 (1992); Nielsen, Nature, 365:566 (1993);Carlsson, et al., Nature, 380:207 (1996), incorporated by reference intheir entireties).

Other analog nucleic acids include those with positive backbones(Denpcy, et al., Proc. Natl. Acad. Sci. USA, 92:6097 (1995),incorporated by reference in its entirety); non-ionic backbones (U.S.Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863;Kiedrowshi, et al., Angew. Chem. Intl. Ed. English, 30:423 (1991);Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); Letsinger, etal., Nucleosides & Nucleotides, 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, et al.,Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996),incorporated by reference in their entireties) and non-ribose (U.S. Pat.Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Coo, incorporated by reference in their entireties).Nucleic acids may also contain one or more carbocyclic sugars (seeJenkins, et al., Chem. Soc. Rev., (1995) pp. 169 176).

Modifications of the ribose-phosphate backbone may be done to facilitatethe addition of additional moieties such as labels, or to increase thestability of such molecules under certain conditions. In addition,mixtures of naturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made. Thenucleic acids may be single stranded or double stranded, as specified,or contain portions of both double stranded or single stranded sequence.The nucleic acid may be DNA, for example, genomic or cDNA, RNA or ahybrid. A nucleic acid can contain any combination of deoxyribo- andribo-nucleotides, and any combination of bases, including uracil,adenine, thymine, cytosine, guanine, inosine, xanthanine,hypoxanthanine, isocytosine, isoguanine, and base analogs such asnitropyrrole (including 3-nitropyrrole) and nitroindole (including5-nitroindole), etc.

In some embodiments, a nucleic acid can include at least one promiscuousbase. Promiscuous bases can base-pair with more than one different typeof base. In some embodiments, a promiscuous base can base-pair with atleast two different types of bases and no more than three differenttypes of bases. An example of a promiscuous base includes inosine thatmay pair with adenine, thymine, or cytosine. Other examples includehypoxanthine, 5-nitroindole, acylic 5-nitroindole, 4-nitropyrazole,4-nitroimidazole and 3-nitropyrrole (Loakes et al., Nucleic Acid Res.22:4039 (1994); Van Aerschot et al., Nucleic Acid Res. 23:4363 (1995);Nichols et al., Nature 369:492 (1994); Berstrom et al., Nucleic AcidRes. 25:1935 (1997); Loakes et al., Nucleic Acid Res. 23:2361 (1995);Loakes et al., J. Mol. Biol. 270:426 (1997); and Fotin et al., NucleicAcid Res. 26:1515 (1998), incorporated by reference in theirentireties). Promiscuous bases that can base-pair with at least three,four or more types of bases can also be used.

As used herein, the term “nucleotide analog” and/or grammaticalequivalents thereof can refer to synthetic analogs having modifiednucleotide base portions, modified pentose portions, and/or modifiedphosphate portions, and, in the case of polynucleotides, modifiedinternucleotide linkages, as generally described elsewhere (e.g.,Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Englisch, Angew.Chem. Int. Ed. Engl. 30:613-29, 1991; Agarwal, Protocols forPolynucleotides and Analogs, Humana Press, 1994; and S. Verma and F.Eckstein, Ann. Rev. Biochem. 67:99-134, 1998). Generally, modifiedphosphate portions comprise analogs of phosphate wherein the phosphorousatom is in the +5 oxidation state and one or more of the oxygen atoms isreplaced with a non-oxygen moiety, e.g., sulfur. Exemplary phosphateanalogs include but are not limited to phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate,boronophosphates, including associated counterions, e.g., H⁺, NH₄ ⁺,Na⁺, if such counterions are present. Example modified nucleotide baseportions include but are not limited to 5-methylcytosine (5mC);C-5-propynyl analogs, including but not limited to, C-5 propynyl-C andC-5 propynyl-U; 2,6-diaminopurine, also known as 2-amino adenine or2-amino-dA); hypoxanthine, pseudouridine, 2-thiopyrimidine, isocytosine(isoC), 5-methyl isoC, and isoguanine (isoG; see, e.g., U.S. Pat. No.5,432,272). Exemplary modified pentose portions include but are notlimited to, locked nucleic acid (LNA) analogs including withoutlimitation Bz-A-LNA, 5-Me-Bz-C-LNA, dmf-G-LNA, and T-LNA (see, e.g., TheGlen Report, 16(2):5, 2003; Koshkin et al., Tetrahedron 54:3607-30,1998), and 2′- or 3′-modifications where the 2′- or 3′-position ishydrogen, hydroxy, alkoxy (e.g., methoxy, ethoxy, allyloxy, isopropoxy,butoxy, isobutoxy and phenoxy), azido, amino, alkylamino, fluoro,chloro, or bromo. Modified internucleotide linkages include phosphateanalogs, analogs having achiral and uncharged intersubunit linkages(e.g., Sterchak, E. P. et al., Organic Chem., 52:4202, 1987), anduncharged morpholino-based polymers having achiral intersubunit linkages(see, e.g., U.S. Pat. No. 5,034,506). Some internucleotide linkageanalogs include morpholidate, acetal, and polyamide-linked heterocycles.In one class of nucleotide analogs, known as peptide nucleic acids,including pseudocomplementary peptide nucleic acids (“PNA”), aconventional sugar and internucleotide linkage has been replaced with a2-aminoethylglycine amide backbone polymer (see, e.g., Nielsen et al.,Science, 254:1497-1500, 1991; Egholm et al., J. Am. Chem. Soc., 114:1895-1897 1992; Demidov et al., Proc. Natl. Acad. Sci. 99:5953-58, 2002;Peptide Nucleic Acids: Protocols and Applications, Nielsen, ed., HorizonBioscience, 2004).

As used herein, the term “sequencing read” and/or grammaticalequivalents thereof can refer to a repetitive process of physical orchemical steps that is carried out to obtain signals indicative of theorder of monomers in a polymer. The signals can be indicative of anorder of monomers at single monomer resolution or lower resolution. Inparticular embodiments, the steps can be initiated on a nucleic acidtarget and carried out to obtain signals indicative of the order ofbases in the nucleic acid target. The process can be carried out to itstypical completion, which is usually defined by the point at whichsignals from the process can no longer distinguish bases of the targetwith a reasonable level of certainty. If desired, completion can occurearlier, for example, once a desired amount of sequence information hasbeen obtained. A sequencing read can be carried out on a single targetnucleic acid molecule or simultaneously on a population of targetnucleic acid molecules having the same sequence, or simultaneously on apopulation of target nucleic acids having different sequences. In someembodiments, a sequencing read is terminated when signals are no longerobtained from one or more target nucleic acid molecules from whichsignal acquisition was initiated. For example, a sequencing read can beinitiated for one or more target nucleic acid molecules that are presenton a solid phase substrate and terminated upon removal of the one ormore target nucleic acid molecules from the substrate. Sequencing can beterminated by otherwise ceasing detection of the target nucleic acidsthat were present on the substrate when the sequencing run wasinitiated.

As used herein, the term “sequencing representation” and/or grammaticalequivalents thereof can refer to information that signifies the orderand type of monomeric units in the polymer. For example, the informationcan indicate the order and type of nucleotides in a nucleic acid. Theinformation can be in any of a variety of formats including, forexample, a depiction, image, electronic medium, series of symbols,series of numbers, series of letters, series of colors, etc. Theinformation can be at single monomer resolution or at lower resolution,as set forth in further detail below. An exemplary polymer is a nucleicacid, such as DNA or RNA, having nucleotide units. A series of “A,” “T,”“G,” and “C” letters is a well known sequence representation for DNAthat can be correlated, at single nucleotide resolution, with the actualsequence of a DNA molecule. Other exemplary polymers are proteins havingamino acid units and polysaccharides having saccharide units.

As used herein the term “at least a portion” and/or grammaticalequivalents thereof can refer to any fraction of a whole amount. Forexample, “at least a portion” can refer to at least about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% of awhole amount.

Transposon Sequences

Some embodiments provided herein include transposon sequences. In someembodiments, a transposon sequence includes at least one transposaserecognition site and at least one barcode. In some embodiments, atransposon sequence includes a first transposon recognition site, asecond transposon recognition site, and a barcode disposed therebetween.FIG. 1 depicts a schematic a transposon sequence.

A transposase recognition site can include two complementary nucleicacid sequences, e.g., a double-stranded nucleic acid or a hairpinnucleic acid, that comprise a substrate for a transposase or integrase.The transposase or integrase may bind to the transposase recognitionsite and insert the transposase recognition site into a target nucleicacid. In some such insertion events, one strand of the transposaserecognition site may be transferred into the target nucleic acid.

In some embodiments a transposase recognition site is a component of atransposition system. A transposition system can include a transposaseenzyme and a transposase recognition site. In some such systems, thetransposase can form a functional complex with a transposes recognitionsite that is capable of catalyzing a transposition reaction. Someembodiments can include the use of a hyperactive Tn5 transposase and aTn5-type transposase recognition site (Goryshin, I. and Reznikoff, W.S., J. Biol. Chem., 273: 7367, 1998), or MuA transposase and a Mutransposase recognition site comprising R1 and R2 end sequences(Mizuuchi, K., Cell, 35: 785, 1983; Savilahti, H, et al., EMBO J., 14:4893, 1995). An exemplary transposase recognition site that forms acomplex with a hyperactive Tn5 transposase (e.g., EZ-Tn5™ Transposase,EPICENTRE Biotechnologies, Madison, Wis., USA) comprises the followingtransferred strand and non-transferred strands: 5′ AGATGTGTATAAGAGACAG3′, (SEQ ID NO: 1), 5′ CTGTCT CTTATACACATCT 3′ (SEQ ID NO: 2),respectively. More examples of transposition systems that can be usedwith certain embodiments provided herein include Staphylococcus aureusTn552 (Colegio O R et al., J. Bacteriol., 183: 2384-8, 2001; Kirby C etal., Mol. Microbiol., 43: 173-86, 2002), Ty1 (Devine S E, and Boeke JD., Nucleic Acids Res., 22: 3765-72, 1994 and International PatentApplication No. WO 95/23875), Transposon Tn7 (Craig, N L, Science. 271:1512, 1996; Craig, N L, Review in: Curr Top Microbiol Immunol., 204:27-48, 1996), Tn/O and IS10 (Kleckner N, et al., Curr Top MicrobiolImmunol., 204: 49-82, 1996), Mariner transposase (Lampe D J, et al.,EMBO J., 15: 5470-9, 1996), Tc1 (Plasterk R H, Curr Top MicrobiolImmunol, 204: 125-43, 1996), P Element (Gloor, G B, Methods Mol. Biol.,260: 97-114, 2004), Tn3 (Ichikawa H, and Ohtsubo E., J. Biol. Chem. 265:18829-32, 1990), bacterial insertion sequences (Ohtsubo, F and Sekine,Y, Curr. Top. Microbiol. Immunol. 204: 1-26, 1996), retroviruses (BrownP O, et al., Proc Natl Acad Sci USA, 86: 2525-9, 1989), andretrotransposon of yeast (Boeke J D and Corces V G, Annu Rev Microbiol.43: 403-34, 1989), the disclosures of which are incorporated herein byreference in their entireties.

Barcodes

Generally, a barcode can include one or more nucleotide sequences thatcan be used to identify one or more particular nucleic acids. A barcodecan comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides. In someembodiments, a barcode comprises at least about 10, 20, 30, 40, 50, 60,70 80, 90, 100 or more consecutive nucleotides. In some embodiments, atleast a portion of the barcodes in a population of nucleic acidscomprising barcodes is different. In some embodiments, at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of the barcodesare different. In more such embodiments, all of the barcodes aredifferent. The diversity of different barcodes in a population ofnucleic acids comprising barcodes can be randomly generated ornon-randomly generated.

In some embodiments, a transposon sequence comprises at least onebarcode. In some embodiments, a transposon sequence comprises a barcodecomprising a first barcode sequence and a second barcode sequence. Insome such embodiments, the first barcode sequence can be identified ordesignated to be paired with the second barcode sequence. For example, aknown first barcode sequence can be known to be paired with a knownsecond barcode sequence using a reference table comprising a pluralityof first and second bar code sequences known to be paired to oneanother. In another example, the first barcode sequence can comprise thesame sequence as the second barcode sequence. In another example, thefirst barcode sequence can comprise the reverse complement of the secondbarcode sequence.

In some embodiments, a population of nucleic acids can comprise nucleicacids that include a first barcode sequence and second barcode sequence.In some such embodiments the first and second barcode sequences of aparticular nucleic acid can be different. As will be described furtherherein, paired first and second barcode sequences can be used toidentify different nucleic acids comprising barcodes linked with oneanother.

Linkers

Some embodiments include transposon sequences comprising a first barcodesequence and a second barcode sequence having a linker disposedtherebetween. In other embodiments, the linker can be absent, or can bethe sugar-phosphate backbone that connects one nucleotide to another.The linker can comprise, for example, one or more of a nucleotide, anucleic acid, a non-nucleotide chemical moiety, a nucleotide analogue,amino acid, peptide, polypeptide, or protein. In preferred embodiments,a linker comprises a nucleic acid. The linker can comprise at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more nucleotides. In some embodiments, a linker can comprise atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or morenucleotides.

Fragmentation Sites

In some embodiments, the linker can comprise a fragmentation site. Afragmentation site can be used to cleave the physical, but not theinformational association between a first barcode sequence and a secondbarcode sequence. Cleavage may be by biochemical, chemical or othermeans. In some embodiments, a fragmentation site can include anucleotide or nucleotide sequence that may be fragmented by variousmeans. For example, a fragmentation site may be a substrate for anenzyme, such as a nuclease, that will cleave the physical associationbetween a first barcode sequence and a second barcode sequence. Forexample, the fragmentation site comprises a restriction endonucleasesite and may be cleaved with an appropriate restriction endonuclease. Inanother example, a fragmentation site can comprise at least oneribonucleotide in a nucleic acid that may otherwise comprisedeoxyribonucleotides and may be cleaved with an RNAse. Chemical cleavageagents capable of selectively cleaving the phosphodiester bond between adeoxyribonucleotide and a ribonucleotide include metal ions, for examplerare-earth metal ions (e.g., La³⁺, particularly Tm³⁺, Yb³⁺ or Lu³⁺ (Chenet al. Biotechniques. 2002, 32: 518-520; Komiyama et al. Chem. Commun.1999, 1443-1451)), Fe(3) or Cu(3), or exposure to elevated pH, e.g.,treatment with a base such as sodium hydroxide. As used herein,selective cleavage of the phosphodiester bond between adeoxyribonucleotide and a ribonucleotide can refer to the chemicalcleavage agent is not capable of cleaving the phosphodiester bondbetween two deoxyribonucleotides under the same conditions.

In another example, the fragmentation site can comprise one or morerecognition sequences for a nickase, that is, a nicking endonucleasethat breaks one strand of a double-stranded nucleic acid. Thus, thefragmentation site can comprise a first nickase recognition sequence, asecond nickase recognition sequence. The cut site for each recognitionsequence can be the same site or different site.

In another example, a fragmentation site can include one or morenucleotide analogues that comprise an abasic site and permits cleavageat the fragmentation site in the presence of certain chemical agents,such as polyamine, N,N′-dimethylethylenediamine (DMED) (U.S. PatentApplication Publication No. 2010/0022403, incorporated by referenceherein). In some embodiments, an abasic site may be created within afragmentation site by first providing a fragmentation site comprising adeoxyuridine (U) of a double stranded nucleic acid. The enzyme uracilDNA glycosylase (UDG) may then be used to remove the uracil base,generating an abasic site on one strand. The polynucleotide strandincluding the abasic site may then be cleaved at the abasic site bytreatment with endonuclease (e.g. Endo IV endonuclease, AP lyase, FPGglycosylase/AP lyase, Endo VIII glycosylase/AP lyase), heat or alkali.Abasic sites may also be generated at nucleotide analogues other thandeoxyuridine and cleaved in an analogous manner by treatment withendonuclease, heat or alkali. For example, 8-oxo-guanine can beconverted to an abasic site by exposure to FPG glycosylase. Deoxyinosinecan be converted to an abasic site by exposure to AlkA glycosylase. Theabasic sites thus generated may then be cleaved, typically by treatmentwith a suitable endonuclease (e.g. Endo IV, AP lyase). (U.S. PatentApplication Publication No. 2011/0014657, incorporated by referenceherein in its entirety).

In another example, a fragmentation site may include a diol linkagewhich permits cleavage by treatment with periodate (e.g., sodiumperiodate). In another example, a fragmentation site may include adisulphide group which permits cleavage with a chemical reducing agent,e.g. Tris (2-carboxyethyl)-phosphate hydrochloride (TCEP).

In some embodiments, a fragmentation site may include a cleavable moietythat may be subject to photochemical cleavage. Photochemical cleavageencompasses any method which utilizes light energy in order to achievecleavage of a nucleic acids, for example, one or both strands of adouble-stranded nucleic acid molecule. A site for photochemical cleavagecan be provided by a non-nucleotide chemical moiety in a nucleic acid,such as phosphoamidite(4-(4,4′-Dimethoxytrityloxy)butyramidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite)(Glen Research, Sterling, Va., USA, Cat No. 10-4913-XX).

In some embodiments, a fragmentation site can include a peptide, forexample, conjugate structure in which a peptide molecule is linked to anucleic acid. The peptide molecule can subsequently be cleaved by apeptidase enzyme of the appropriate specificity, or any other suitablemeans of non-enzymatic chemical or photochemical cleavage. In someembodiments, a conjugate between peptide and nucleic acid will be formedby covalently linking a peptide to a nucleic acid, e.g., a strand of adouble-stranded nucleic acid. Conjugates between a peptide and nucleicacid can be prepared using techniques generally known in the art. In onesuch technique the peptide and nucleic acid components of the desiredamino acid and nucleotide sequence can be synthesized separately, e.g.by standard automated chemical synthesis techniques, and then conjugatedin aqueous/organic solution. By way of example, the OPeC™ systemcommercially available from Glen Research is based on the nativeligation of an N-terminal thioester-functionalized peptide to a5′-cysteinyl oligonucleotide.

Primer Sites

In some embodiments, a linker can be a “sequencing adaptor” or“sequencing adaptor site”, that is to say a region that comprises one ormore sites that can hybridize to a primer. In some embodiments, a linkercomprises at least a first primer site. In some embodiments, a linkercomprises at least a first primer site and a second primer site. Theorientation of the primer sites in such embodiments can be such that aprimer hybridizing to the first primer site and a primer hybridizing tothe second primer site are in the same orientation, or in differentorientations. In one embodiment, the primer sequence in the linker canbe complementary to a primer used for amplification. In anotherembodiment, the primer sequence is complementary to a primer used forsequencing.

In some embodiments, a linker can include a first primer site, a secondprimer site having a non-amplifiable site disposed therebetween. Thenon-amplifiable site is useful to block extension of a polynucleotidestrand between the first and second primer sites, wherein thepolynucleotide strand hybridizes to one of the primer sites. Thenon-amplifiable site can also be useful to prevent concatamers. Examplesof non-amplifiable sites include a nucleotide analogue, non-nucleotidechemical moiety, amino-acid, peptide, and polypeptide. In someembodiments, a non-amplifiable site comprises a nucleotide analogue thatdoes not significantly basepair with A, C, G or T.

Some embodiments include a linker comprising a first primer site, asecond primer site having a fragmentation site disposed therebetween.

Other embodiments can use a forked or Y-shaped adapter design useful fordirectional sequencing, as described in U.S. Pat. No. 7,741,463, whichis incorporated herein by reference, An example is shown in FIG. 12.

Affinity Tags

In some embodiments, a linker can comprise an affinity tag. Affinitytags can be useful for the bulk separation of target nucleic acidshybridized to hybridization tags. As used herein, the term “affinitytag” and grammatical equivalents can refer to a component of amulti-component complex, wherein the components of the multi-componentcomplex specifically interact with or bind to each other. For example anaffinity tag can include biotin or His that can bind streptavidin ornickel, respectively. Other examples of multiple-component affinity tagcomplexes include, ligands and their receptors, for example,avidin-biotin, streptavidin-biotin, and derivatives of biotin,streptavidin, or avidin, including, but not limited to, 2-iminobiotin,desthiobiotin, NeutrAvidin (Molecular Probes, Eugene, Oreg.), CaptAvidin(Molecular Probes), and the like; binding proteins/peptides, includingmaltose-maltose binding protein (MBP), calcium-calcium bindingprotein/peptide (CBP); antigen-antibody, including epitope tags,including c-MYC (e.g., EQKLISEEDL (SEQ ID NO: 3)), HA (e.g., YPYDVPDYA(SEQ ID NO: 4)), VSV-G (e.g., YTDIEMNRLGK (SEQ ID NO: 5)), HSV (e.g.,QPELAPEDPED (SEQ ID NO: 6)), V5 (e.g., GKPIPNPLLGLDST (SEQ ID NO: 7)),and FLAG Tag™. (e.g., DYKDDDDKG (SEQ ID NO: 8)), and their correspondinganti-epitope antibodies; haptens, for example, dinitrophenyl anddigoxigenin, and their corresponding antibodies; aptamers and theircorresponding targets; poly-His tags (e.g., penta-His and hexa-His) andtheir binding partners including corresponding immobilized metal ionaffinity chromatography (IMAC) materials and anti-poly-His antibodies;fluorophores and anti-fluorophore antibodies; and the like.

Reporter Moieties

In some embodiments, a linker can comprise a reporter moiety. As usedherein, the term “reporter moiety” and grammatical equivalents can referto any identifiable tag, label, or group. The skilled artisan willappreciate that many different species of reporter moieties can be usedwith the methods and compositions described herein, either individuallyor in combination with one or more different reporter moieties. Incertain embodiments, a reporter moiety can emit a signal. Examples ofsignals fluorescent, a chemiluminescent, a bioluminescent, aphosphorescent, a radioactive, a calorimetric, or anelectrochemiluminescent signals. Example reporter moieties includefluorophores, radioisotopes, chromogens, enzymes, antigens includingepitope tags, semiconductor nanocrystals such as quantum dots, heavymetals, dyes, phosphorescence groups, chemiluminescent groups,electrochemical detection moieties, binding proteins, phosphors, rareearth chelates, transition metal chelates, near-infrared dyes,electrochemiluminescence labels, and mass spectrometer compatiblereporter moieties, such as mass tags, charge tags, and isotopes. Morereporter moieties that may be used with the methods and compositionsdescribed herein include spectral labels such as fluorescent dyes (e.g.,fluorescein isothiocyanate, Texas red, rhodamine, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.), enzymes (e.g.,horse-radish peroxidase, alkaline phosphatase etc.) spectralcalorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads; magnetic,electrical, thermal labels; and mass tags. Reporter moieties can alsoinclude enzymes (horseradish peroxidase, etc.) and magnetic particles.More reporter moieties include chromophores, phosphors and fluorescentmoieties, for example, Texas red, dixogenin, biotin, 1- and2-aminonaphthalene, p,p′-diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p′-diaminobenzophenone imines,anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene,bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthen,7-hydroxycoumarin, phenoxazine, calicylate, strophanthidin, porphyrins,triarylmethanes and flavin. Individual fluorescent compounds which havefunctionalities for linking to an element desirably detected in anapparatus or assay provided herein, or which can be modified toincorporate such functionalities include, e.g., dansyl chloride;fluoresceins such as 3,6-dihydroxy-9-phenylxanthydrol;rhodamineisothiocyanate; N-phenyl 1-amino-8-sulfonatonaphthalene;N-phenyl 2-amino-6-sulfonatonaphthalene;4-acetamido-4-isothiocyanato-stilbene-2,2′-disulfonic acid;pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate;N-phenyl-N-methyl-2-aminoaphthalene-6-sulfonate; ethidium bromide;stebrine; auromine-0,2-(9′-anthroyl)palmitate; dansylphosphatidylethanolamine; N,N′-dioctadecyl oxacarbocyanine: N,N′-dihexyloxacarbocyanine; merocyanine, 4-(3′-pyrenyl)stearate;d-3-aminodesoxy-equilenin; 12-(9′-anthroyl)stearate; 2-methylanthracene;9-vinylanthracene; 2,2′(vinylene-p-phenylene)bisbenzoxazole;p-bis(2-methyl-5-phenyl-oxazolyl))benzene;6-dimethylamino-1,2-benzophenazin; retinol; bis(3′-aminopyridinium)1,10-decandiyl diiodide; sulfonaphthylhydrazone of hellibrienin;chlorotetracycline;N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide;N-(p-(2-benzimidazolyl)-phenyl)maleimide; N-(4-fluoranthyl)maleimide;bis(homovanillic acid); resazarin;4-chloro7-nitro-2,1,3-benzooxadiazole; merocyanine 540; resorufin; rosebengal; 2,4-diphenyl-3(2H)-furanone, fluorescent lanthanide complexes,including those of Europium and Terbium, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,quantum dots (also referred to as “nanocrystals”: see U.S. Pat. No.6,544,732, hereby incorporated by reference), pyrene, Malacite green,stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, Cy dyes (Cy3, Cy5,etc.), alexa dyes, phycoerythin, bodipy, and others described in the 6thEdition of the Molecular Probes Handbook by Richard P. Haugland,expressly incorporated by reference herein.

Certain Methods of Making Transposon Sequences

The transposon sequences provided herein can be prepared by a variety ofmethods. Exemplary methods include direct synthesis and hairpinextension methods. In some embodiments, transposon sequences may beprepared by direct synthesis. For example, a transposon sequencecomprising a nucleic acid may be prepared by methods comprising chemicalsynthesis. Such methods are well known in the art, e.g., solid phasesynthesis using phosphoramidite precursors such as those derived fromprotected 2′-deoxynucleosides, ribonucleosides, or nucleoside analogues.

In some embodiments, a transposon sequence can be prepared by hairpinextension. In some such embodiments, a portion of a transposon sequencemay be prepared by chemical synthesis and extended by hairpin extension.In an example embodiment, a precursor transposon sequence comprising apolynucleotide may include a first transposase recognition site, and afirst barcode sequence. The precursor transposon sequence may beextended using an appropriate nucleic acid polymerase by hairpinextension, thereby preparing a hairpin structure comprising a firsttransposase recognition site, a first barcode sequence, a second barcodesequence, and a second transposase recognition site. In such atransposon sequence, the first transposase recognition site and firstbarcode sequence can have the reverse complement sequence of, and thesecond transposase recognition site and the second barcode sequence,respectively.

Some methods of preparing transposons sequences can include preparingbarcode sequences. Barcode sequences can be generated randomly andnon-randomly. In some embodiments, at least a portion of a barcodesequence can be generated randomly. In some embodiments a barcodesequence can be generated using combinatorial methods. In some suchmethods, barcode sequences can comprise one or more subunits comprisingone or more consecutive nucleotides. As will be understood, in someembodiments, a subunit can comprise a nucleotide analogue and/or anucleotide comprising a label. At least a portion of a barcode sequencecan comprise at least one subunit. Barcode sequences comprising at leastone subunit can be generated randomly or non-randomly. In someembodiments, at least a portion of a barcode sequences comprising atleast one subunit can be generated randomly or non-randomly. As will beunderstood, in some embodiments, at least a portion of a barcodesequence is known.

Primer sites may be ligated to the ends of the hairpin structure inorder to generate a complementary strand to the single-strand of thehairpin structure. An exemplary embodiment of a method of making atransposon sequence is depicted in FIG. 2. It will be understood thatthe primer sites introduced to generate the complementary strand may beremoved by a transposase during a transposition reaction.

Target Nucleic Acids

A target nucleic acid can include any nucleic acid of interest. Targetnucleic acids can include, DNA, RNA, peptide nucleic acid, morpholinonucleic acid, locked nucleic acid, glycol nucleic acid, threose nucleicacid, mixtures thereof, and hybrids thereof. In a preferred embodiment,genomic DNA fragments or amplified copies thereof are used as the targetnucleic acid. In another preferred embodiment, mitochondrial orchloroplast DNA is used.

A target nucleic acid can comprise any nucleotide sequence. In someembodiments, the target nucleic acid comprises homopolymer sequences. Atarget nucleic acid can also include repeat sequences. Repeat sequencescan be any of a variety of lengths including, for example, 2, 5, 10, 20,30, 40, 50, 100, 250, 500, 1000 nucleotides or more. Repeat sequencescan be repeated, either contiguously or non-contiguously, any of avariety of times including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20 times or more.

Some embodiments described herein can utilize a single target nucleicacid. Other embodiments can utilize a plurality of target nucleic acids.In such embodiments, a plurality of target nucleic acids can include aplurality of the same target nucleic acids, a plurality of differenttarget nucleic acids where some target nucleic acids are the same, or aplurality of target nucleic acids where all target nucleic acids aredifferent. Embodiments that utilize a plurality of target nucleic acidscan be carried out in multiplex formats such that reagents are deliveredsimultaneously to the target nucleic acids, for example, in a one ormore chambers or on an array surface. In some embodiments, the pluralityof target nucleic acids can include substantially all of a particularorganism's genome. The plurality of target nucleic acids can include atleast a portion of a particular organism's genome including, forexample, at least about 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%,or 99% of the genome. In particular embodiments the portion can have anupper limit that is at most about 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%,90%, 95%, or 99% of the genome

Target nucleic acids can be obtained from any source. For example,target nucleic acids may be prepared from nucleic acid moleculesobtained from a single organism or from populations of nucleic acidmolecules obtained from natural sources that include one or moreorganisms. Sources of nucleic acid molecules include, but are notlimited to, organelles, cells, tissues, organs, or organisms. Cells thatmay be used as sources of target nucleic acid molecules may beprokaryotic (bacterial cells, for example, Escherichia, Bacillus,Serratia, Salmonella, Staphylococcus, Streptococcus, Clostridium,Chlamydia, Neisseria, Treponema, Mycoplasma, Borrelia, Legionella,Pseudomonas, Mycobacterium, Helicobacter, Erwinia, Agrobacterium,Rhizobium, and Streptomyces genera); archeaon, such as crenarchaeota,nanoarchaeota or euryarchaeotia; or eukaryotic such as fungi, (forexample, yeasts), plants, protozoans and other parasites, and animals(including insects (for example, Drosophila spp.), nematodes (forexample, Caenorhabditis elegans), and mammals (for example, rat, mouse,monkey, non-human primate and human)).

Methods of Preparing Template Nucleic Acids

Some embodiments include methods of preparing template nucleic acids. Asused herein, the term “template nucleic acid” can refer to a targetnucleic acid, a fragment thereof, or any copy thereof comprising atleast one transposon sequence, a fragment thereof, or any copy thereof.Accordingly, some methods of preparing template nucleic acids includeinserting a transposon sequence into a target nucleic acid, therebypreparing a template nucleic acid. Some methods of insertion includecontacting a transposon sequence provided herein with a target nucleicacid in the presence of an enzyme, such as a transposase or integrase,under conditions sufficient for the integration of the transposonsequence into the target nucleic acid.

Exemplary transposition systems that may be utilized with thecompositions and methods provided herein include a hyperactive Tn5transposase and a Tn5-type transposase recognition site (Goryshin, I.and Reznikoff, W. S., J. Biol. Chem., 273: 7367, 1998; US Pub.2010/0120098, which is incorporated herein by reference), and MuAtransposase and a Mu transposase recognition site comprising R1 and R2end sequences (Mizuuchi, K., Cell, 35: 785, 1983; Savilahti, H, et al.,EMBO J., 14: 4893, 1995). More examples include sequences and enzymesrelated to Staphylococcus aureus Tn552 (Colegio O R et al., J.Bacteriol., 183: 2384-8, 2001; Kirby C et al., Mol. Microbiol., 43:173-86, 2002), Ty1 (Devine S E, and Boeke J D., Nucleic Acids Res., 22:3765-72, 1994 and International Patent Application No. WO 95/23875),Transposon Tn7 (Craig, N L, Science. 271: 1512, 1996; Craig, N L, Reviewin: Curr Top Microbiol Immunol., 204: 27-48, 1996), Tn/O and IS10(Kleckner N, et al., Curr Top Microbiol Immunol., 204: 49-82, 1996),Mariner transposase (Lampe D J, et al., EMBO J., 15: 5470-9, 1996), Tc1(Plasterk R H, Curr Top Microbiol Immunol, 204: 125-43, 1996), P Element(Gloor, G B, Methods Mol. Biol., 260: 97-114, 2004), Tn3 (Ichikawa H,and Ohtsubo E., J. Biol. Chem. 265: 18829-32, 1990), bacterial insertionsequences (Ohtsubo, F and Sekine, Y, Curr. Top. Microbiol. Immunol. 204:1-26, 1996), retroviruses (Brown P O, et al., Proc Natl Acad Sci USA,86: 2525-9, 1989), and retrotransposon of yeast (Boeke J D and Corces VG, Annu Rev Microbiol. 43: 403-34, 1989).

In some embodiments, insertion of transposon sequences into a targetnucleic acid can be non-random. In some embodiments, transposonsequences can be contacted with target nucleic acids comprising proteinsthat inhibit integration at certain sites. For example, transposonsequences can be inhibited from integrating into genomic DNA comprisingproteins, genomic DNA comprising chromatin, genomic DNA comprisingnucleosomes, or genomic DNA comprising histones.

It will be understood that during integration of some transposonsequences into a target nucleic acid, several consecutive nucleotides ofthe target nucleic acid at the integration site are duplicated in theintegrated product. Thus the integrated product can include a duplicatedsequence at each end of the integrated sequence in the target nucleicacid. An example of such a duplication event is depicted in FIG. 3. Asused herein, the term “host tag” can refer to a target nucleic acidsequence that is duplicated at each end of an integrated transposonsequence.

In some embodiments, a plurality of the transposon sequences providedherein is inserted into a target nucleic acid. Some embodiments includeselecting conditions sufficient to achieve integration of a plurality oftransposon sequences into a target nucleic acid such that the averagedistance between each integrated transposon sequence comprises a certainnumber of consecutive nucleotides in the target nucleic acid.

In some embodiments, conditions may be selected so that the averagedistance in a target nucleic acid between integrated transposonsequences is at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,or more consecutive nucleotides. In some embodiments, the averagedistance in a target nucleic acid between integrated transposonsequences is at least about 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, or more consecutive nucleotides. In some embodiments, the averagedistance in a target nucleic acid between integrated transposonsequences is at least about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8kb, 90 kb, 100 kb, or more consecutive nucleotides. In some embodiments,the average distance in a target nucleic acid between integratedtransposon sequences is at least about 100 kb, 200 kb, 300 kb, 400 kb,500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, or more consecutivenucleotides. As will be understood, some conditions that may be selectedinclude contacting a target nucleic acid with a certain number oftransposon sequences.

Some embodiments include selecting conditions sufficient to achieve atleast a portion of transposon sequences integrated into a target nucleicacid are different. In preferred embodiments, each transposon sequenceintegrated into a target nucleic acid is different. Some conditions thatmay be selected to achieve a certain portion of transposon sequencesintegrated into a target sequences that are different include selectingthe degree of diversity of the population of transposon sequences. Aswill be understood, the diversity of transposon sequences arises in partdue to the diversity of the barcodes of such transposon sequences.Accordingly, some embodiments include providing a population oftransposon sequences in which at least a portion of the barcodes aredifferent. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of barcodes in a populationof transposon sequences are different.

Some embodiments of preparing a template nucleic acid can includecopying the sequences comprising the target nucleic acid. For example,some embodiments include hybridizing a primer to a primer site of atransposon sequence integrated into the target nucleic acid. In somesuch embodiments, the primer can be hybridized to the primer site andextended. The copied sequences can include at least one barcode sequenceand at least a portion of the target nucleic acid. In some embodiments,the copied sequences can include a first barcode sequence, a secondbarcode sequence, and at least a portion of a target nucleic aciddisposed therebetween. In some embodiments, at least one copied nucleicacid can include at least a first barcode sequence of a first copiednucleic acid that can be identified or designated to be paired with asecond barcode sequence of a second copied nucleic acid. In someembodiments, the primer can include a sequencing primer. In someembodiments sequencing data is obtained using the sequencing primer.

Some embodiments of preparing a template nucleic acid can includeamplifying sequences comprising at least a portion of one or moretransposon sequences and at least a portion of a target nucleic acid. Insome embodiments, at least a portion of a target nucleic acid can beamplified using primers that hybridize to primer sites of integratedtransposon sequences integrated into a target nucleic acid. In some suchembodiments, an amplified nucleic acid can include a first barcodesequence, and second barcode sequence having at least a portion of thetarget nucleic acid disposed therebetween. In some embodiments, at leastone amplified nucleic acid can include at least a first barcode sequenceof a first amplified nucleic acid that can be identified to be pairedwith a second barcode sequence of a second amplified sequence.

Some embodiments of preparing a template nucleic acid can includefragmenting a target nucleic acid comprising transposon sequences.Methods of fragmenting nucleic acids are well known in the art. In someembodiments, a nucleic acid comprising transposon sequences can befragmented at random positions along the length of the nucleic acid. Insome embodiments, a target nucleic acid comprising transposon sequencescan be fragmented at the fragmentation sites of the transposonsequences.

Further embodiments of preparing a template nucleic acid that includefragmenting a target nucleic acid comprising transposon sequences canalso include amplifying the fragmented nucleic acids. In someembodiments, the fragmented nucleic acids can be amplified using primersthat hybridize to primer sites of transposon sequences. In moreembodiments, primer sites can be ligated to the ends of the fragmentednucleic acids. In some such embodiments, the fragmented nucleic acidswith ligated primer sites can be amplified from such primer sites.

Some embodiments include reducing the complexity of a library oftemplate nucleic acids. A complexity-reduction step can be performedbefore or after the fragmentation step in the method. For example, thetarget nucleic acid comprising the transposon sequences can be dilutedso that a small number or a single molecule represents the targetdiluted before performing subsequent steps.

FIG. 4 depicts an exemplary embodiment of a contacting a target nucleicacid with a library of transposons, which integrates into the targetnucleic acid. FIG. 5 depicts an optional step of fragmenting a linkerinto two parts, or an optional step of virtually fragmenting the linkerby amplification using a primer A′ and/or a primer B′. Fragmentation ofthe linker generates a library of template nucleic acids. In thisfigure, the filled-in region is indicated by a thick line.

Methods of Analyzing Template Nucleic Acids

Some embodiments include methods of analyzing template nucleic acids.Sequencing information can be obtained from a template nucleic acids anda sequence representation of the target nucleic acid can be obtainedfrom such sequencing data.

In some embodiments, a linked read strategy may be used. A linked readstrategy can include identifying sequencing data that links at least twosequencing reads. For example, a first sequencing read may contain afirst marker, and a second sequencing read may contain a second marker.The first and second markers can identify the sequencing data from eachsequencing read to be adjacent in a sequence representation of thetarget nucleic acid. In some embodiments, markers can comprise a firstbarcode sequence and a second barcode sequence in which the firstbarcode sequence can be paired with the second barcode sequence. In moreembodiments, markers can comprise a first host tag and a second hosttag. In more embodiments, markers can comprise a first barcode sequencewith a first host tag, and a second barcode sequence with a second hosttag.

An exemplary embodiment of a method for sequencing a template nucleicacid can comprise the following steps. First, sequence the first barcodesequence using a primer hybridizing to the first primer site as thesequencing primer; second, sequence the second barcode sequence using aprimer hybridizing to the second primer site as the sequencing primer.The result is two sequence reads that help link the read to its genomicneighbors. Given long enough reads, and short enough library fragments,these two reads can be merged informatically to make one long read thatcovers the entire fragment. Using the barcode sequence reads and the 9nucleotide duplicated sequence present from the insertion, reads can nowbe linked to their genomic neighbors to form much longer “linked reads”in silico.

FIG. 6 depicts an exemplary embodiment of a method for assembling shortsequencing reads using a linked read strategy. In such an exemplaryembodiment, transposon sequences comprising barcodes are inserted intogenomic DNA, a library is prepared and sequencing data obtained for thelibrary of template nucleic acids. Blocks of templates are assembled byidentifying paired barcodes and larger contigs are then assembled. FIG.8, FIG. 9 and FIG. 10 depict exemplary embodiments of methods ofassembling sequencing reads using a linked ready strategy.

Sequencing Methods

The methods described herein can be used in conjunction with a varietyof sequencing techniques. In some embodiments, the process to determinethe nucleotide sequence of a target nucleic acid can be an automatedprocess.

Some embodiments include pyrosequencing techniques. Pyrosequencingdetects the release of inorganic pyrophosphate (PPi) as particularnucleotides are incorporated into the nascent strand (Ronaghi, M.,Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996)“Real-time DNA sequencing using detection of pyrophosphate release.”Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencingsheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M.,Uhlen, M. and Nyren, P. (1998) “A sequencing method based on real-timepyrophosphate.” Science 281(5375), 363; U.S. Pat. No. 6,210,891; U.S.Pat. No. 6,258,568 and U.S. Pat. No. 6,274,320, the disclosures of whichare incorporated herein by reference in their entireties). Inpyrosequencing, released PPi can be detected by being immediatelyconverted to adenosine triphosphate (ATP) by ATP sulfurylase, and thelevel of ATP generated is detected via luciferase-produced photons.

In another example type of SBS, cycle sequencing is accomplished bystepwise addition of reversible terminator nucleotides containing, forexample, a cleavable or photobleachable dye label as described, forexample, in U.S. Pat. No. 7,427,67, U.S. Pat. No. 7,414,1163 and U.S.Pat. No. 7,057,026, the disclosures of which are incorporated herein byreference. This approach is being commercialized by Solexa (now IlluminaInc.), and is also described in WO 91/06678 and WO 07/123,744 (filed inthe United States patent and trademark Office as U.S. Ser. No.12/295,337), each of which is incorporated herein by reference in theirentireties. The availability of fluorescently-labeled terminators inwhich both the termination can be reversed and the fluorescent labelcleaved facilitates efficient cyclic reversible termination (CRT)sequencing. Polymerases can also be co-engineered to efficientlyincorporate and extend from these modified nucleotides.

Additional example SBS systems and methods which can be utilized withthe methods and systems described herein are described in U.S. PatentApplication Publication No. 2007/0166705, U.S. Patent ApplicationPublication No. 2006/0188901, U.S. Pat. No. 7,057,026, U.S. PatentApplication Publication No. 2006/0240439, U.S. Patent ApplicationPublication No. 2006/0281109, PCT Publication No. WO 05/065814, U.S.Patent Application Publication No. 2005/0100900, PCT Publication No. WO06/064199 and PCT Publication No. WO 07/010,251, the disclosures ofwhich are incorporated herein by reference in their entireties.

Some embodiments can utilize sequencing by ligation techniques. Suchtechniques utilize DNA ligase to incorporate nucleotides and identifythe incorporation of such nucleotides. Example SBS systems and methodswhich can be utilized with the methods and systems described herein aredescribed in U.S. Pat. No. 6,969,488, U.S. Pat. No. 6,172,218, and U.S.Pat. No. 6,306,597, the disclosures of which are incorporated herein byreference in their entireties.

Some embodiments can include techniques such as next-next technologies.One example can include nanopore sequencing techniques (Deamer, D. W. &Akeson, M. “Nanopores and nucleic acids: prospects for ultrarapidsequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D.Branton, “Characterization of nucleic acids by nanopore analysis”. Acc.Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin,and J. A. Golovchenko, “DNA molecules and configurations in asolid-state nanopore microscope” Nat. Mater. 2:611-615 (2003), thedisclosures of which are incorporated herein by reference in theirentireties). In such embodiments, the target nucleic acid passes througha nanopore. The nanopore can be a synthetic pore or biological membraneprotein, such as α-hemolysin. As the target nucleic acid passes throughthe nanopore, each base-pair can be identified by measuring fluctuationsin the electrical conductance of the pore. (U.S. Pat. No. 7,001,792;Soni, G. V. & Meller, “A. Progress toward ultrafast DNA sequencing usingsolid-state nanopores.” Clin. Chem. 53, 1996-2001 (2007); Healy, K.“Nanopore-based single-molecule DNA analysis.” Nanomed. 2, 459-481(2007); Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. “Asingle-molecule nanopore device detects DNA polymerase activity withsingle-nucleotide resolution.” J. Am. Chem. Soc. 130, 818-820 (2008),the disclosures of which are incorporated herein by reference in theirentireties). In some such embodiments, nanopore sequencing techniquescan be useful to confirm sequence information generated by the methodsdescribed herein.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. Nucleotide incorporations can be detectedthrough fluorescence resonance energy transfer (FRET) interactionsbetween a fluorophore-bearing polymerase and γ-phosphate-labelednucleotides as described, for example, in U.S. Pat. No. 7,329,492 andU.S. Pat. No. 7,211,414 (each of which is incorporated herein byreference in their entireties) or nucleotide incorporations can bedetected with zero-mode waveguides as described, for example, in U.S.Pat. No. 7,315,019 (which is incorporated herein by reference in itsentirety) and using fluorescent nucleotide analogs and engineeredpolymerases as described, for example, in U.S. Pat. No. 7,405,281 andU.S. Patent Application Publication No. 2008/0108082 (each of which isincorporated herein by reference in their entireties). The illuminationcan be restricted to a zeptoliter-scale volume around a surface-tetheredpolymerase such that incorporation of fluorescently labeled nucleotidescan be observed with low background (Levene, M. J. et al. “Zero-modewaveguides for single-molecule analysis at high concentrations.” Science299, 682-686 (2003); Lundquist, P. M. et al. “Parallel confocaldetection of single molecules in real time.” Opt. Lett. 33, 1026-1028(2008); Korlach, J. et al. “Selective aluminum passivation for targetedimmobilization of single DNA polymerase molecules in zero-mode waveguidenanostructures.” Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), thedisclosures of which are incorporated herein by reference in theirentireties). In one example single molecule, real-time (SMRT) DNAsequencing technology provided by Pacific Biosciences Inc can beutilized with the methods described herein. In some embodiments, a SMRTchip or the like may be utilized (U.S. Pat. Nos. 7,181,122, 7,302,146,7,313,308, incorporated by reference in their entireties). A SMRT chipcomprises a plurality of zero-mode waveguides (ZMW). Each ZMW comprisesa cylindrical hole tens of nanometers in diameter perforating a thinmetal film supported by a transparent substrate. When the ZMW isilluminated through the transparent substrate, attenuated light maypenetrate the lower 20-30 nm of each ZMW creating a detection volume ofabout 1×10⁻²¹ L. Smaller detection volumes increase the sensitivity ofdetecting fluorescent signals by reducing the amount of background thatcan be observed.

SMRT chips and similar technology can be used in association withnucleotide monomers fluorescently labeled on the terminal phosphate ofthe nucleotide (Korlach J. et al., “Long, processive enzymatic DNAsynthesis using 100% dye-labeled terminal phosphate-linked nucleotides.”Nucleosides, Nucleotides and Nucleic Acids, 27:1072-1083, 2008;incorporated by reference in its entirety). The label is cleaved fromthe nucleotide monomer on incorporation of the nucleotide into thepolynucleotide. Accordingly, the label is not incorporated into thepolynucleotide, increasing the signal:background ratio. Moreover, theneed for conditions to cleave a label from a labeled nucleotide monomersis reduced.

An additional example of a sequencing platform that may be used inassociation with some of the embodiments described herein is provided byHelicos Biosciences Corp. In some embodiments, TRUE SINGLE MOLECULESEQUENCING can be utilized (Harris T. D. et al., “Single Molecule DNASequencing of a viral Genome” Science 320:106-109 (2008), incorporatedby reference in its entirety). In one embodiment, a library of targetnucleic acids can be prepared by the addition of a 3′ poly(A) tail toeach target nucleic acid. The poly(A) tail hybridizes to poly(T)oligonucleotides anchored on a glass cover slip. The poly(T)oligonucleotide can be used as a primer for the extension of apolynucleotide complementary to the target nucleic acid. In oneembodiment, fluorescently-labeled nucleotide monomer, namely, A, C, G,or T, are delivered one at a time to the target nucleic acid in thepresence DNA polymerase. Incorporation of a labeled nucleotide into thepolynucleotide complementary to the target nucleic acid is detected, andthe position of the fluorescent signal on the glass cover slip indicatesthe molecule that has been extended. The fluorescent label is removedbefore the next nucleotide is added to continue the sequencing cycle.Tracking nucleotide incorporation in each polynucleotide strand canprovide sequence information for each individual target nucleic acid.

An additional example of a sequencing platform that can be used inassociation with the methods described herein is provided by CompleteGenomics Inc. Libraries of target nucleic acids can be prepared wheretarget nucleic acid sequences are interspersed approximately every 20 bpwith adaptor sequences. The target nucleic acids can be amplified usingrolling circle replication, and the amplified target nucleic acids canbe used to prepare an array of target nucleic acids. Methods ofsequencing such arrays include sequencing by ligation, in particular,sequencing by combinatorial probe-anchor ligation (cPAL).

In some embodiments using cPAL, about 10 contiguous bases adjacent to anadaptor may be determined. A pool of probes that includes four distinctlabels for each base (A, C, T, G) is used to read the positions adjacentto each adaptor. A separate pool is used to read each position. A poolof probes and an anchor specific to a particular adaptor is delivered tothe target nucleic acid in the presence of ligase. The anchor hybridizesto the adaptor, and a probe hybridizes to the target nucleic acidadjacent to the adaptor. The anchor and probe are ligated to oneanother. The hybridization is detected and the anchor-probe complex isremoved. A different anchor and pool of probes is delivered to thetarget nucleic acid in the presence of ligase.

The sequencing methods described herein can be advantageously carriedout in multiplex formats such that multiple different target nucleicacids are manipulated simultaneously. In particular embodiments,different target nucleic acids can be treated in a common reactionvessel or on a surface of a particular substrate. This allows convenientdelivery of sequencing reagents, removal of unreacted reagents anddetection of incorporation events in a multiplex manner. In embodimentsusing surface-bound target nucleic acids, the target nucleic acids canbe in an array format. In an array format, the target nucleic acids canbe typically coupled to a surface in a spatially distinguishable manner.For example, the target nucleic acids can be bound by direct covalentattachment, attachment to a bead or other particle or associated with apolymerase or other molecule that is attached to the surface. The arraycan include a single copy of a target nucleic acid at each site (alsoreferred to as a feature) or multiple copies having the same sequencecan be present at each site or feature. Multiple copies can be producedby amplification methods such as, bridge amplification or emulsion PCRas described in further detail herein.

The methods set forth herein can use arrays having features at any of avariety of densities including, for example, at least about 10features/cm², 100 features/cm², 500 features/cm², 1,000 features/cm²,5,000 features/cm², 10,000 features/cm², 50,000 features/cm², 100,000features/cm², 1,000,000 features/cm², 5,000,000 features/cm², 10⁷features/cm², 5×10⁷ features/cm², 10⁸ features/cm², 5×10⁸ features/cm²,10⁹ features/cm², 5×10⁹ features/cm², or higher.

Surfaces

In some embodiments, the nucleic acid template provided herein can beattached to a solid support (“substrate”). Substrates can be two- orthree-dimensional and can comprise a planar surface (e.g., a glassslide) or can be shaped. A substrate can include glass (e.g., controlledpore glass (CPG)), quartz, plastic (such as polystyrene (lowcross-linked and high cross-linked polystyrene), polycarbonate,polypropylene and poly(methymethacrylate)), acrylic copolymer,polyamide, silicon, metal (e.g., alkanethiolate-derivatized gold),cellulose, nylon, latex, dextran, gel matrix (e.g., silica gel),polyacrolein, or composites.

Suitable three-dimensional substrates include, for example, spheres,microparticles, beads, membranes, slides, plates, micromachined chips,tubes (e.g., capillary tubes), microwells, microfluidic devices,channels, filters, or any other structure suitable for anchoring anucleic acid. Substrates can include planar arrays or matrices capableof having regions that include populations of template nucleic acids orprimers. Examples include nucleoside-derivatized CPG and polystyreneslides; derivatized magnetic slides; polystyrene grafted withpolyethylene glycol, and the like.

Various methods can be used to attach, anchor or immobilize nucleicacids to the surface of the substrate. The immobilization can beachieved through direct or indirect bonding to the surface. The bondingcan be by covalent linkage. See, Joos et al. (1997) AnalyticalBiochemistry, 247:96-101; Oroskar et al. (1996) Clin. Chem.,42:1547-1555; and Khandjian (1986) Mol. Bio. Rep., 11:107-11. Apreferred attachment is direct amine bonding of a terminal nucleotide ofthe template or the primer to an epoxide integrated on the surface. Thebonding also can be through non-covalent linkage. For example,biotin-streptavidin (Taylor et al. (1991) J. Phys. D: Appl. Phys.,24:1443) and digoxigenin with anti-digoxigenin (Smith et al. (1992)Science, 253:1122, are common tools for anchoring nucleic acids tosurfaces and parallels. Alternatively, the attachment can be achieved byanchoring a hydrophobic chain into a lipid monolayer or bilayer. Othermethods known in the art for attaching nucleic acid molecules tosubstrates can also be used.

Example Applications

Some embodiments include the use of the compositions and methodsprovided herein in single cell applications. In some embodiments, atransposon sequence can be associated with a bead via a cleavablelinker. A single cell, bead comprising the transposon sequence, andtransposase are provided in a droplet. The cell is lysed and thetransposon sequence cleaved form the bead. The transposition reaction isintiated. Such methods can be used to provide a library of templatenucleic acids for each cell in population.

The following Examples provide illustrative embodiments and do not inany way limit the inventions provided herein.

EXAMPLES Example 1 Whole Genome Amplification Using Transposon Sequences

This example illustrates a method for uniform amplification of genomicDNA with random insertion therein of specific primer sites. Transposonsequences are prepared, each comprising a first transposase recognitionsite, a second transposase recognition site having a sequencing adaptordisposed therebetween, in which the sequencing adaptor comprises a firstprimer site and second primer site. The transposon sequences arecontacted with genomic DNA in the presence of MuA transposase underconditions sufficient for the transposon sequences to integrate into thegenomic DNA. The genomic DNA is amplified using primers that hybridizeto the first primer site or second primer site.

Example 2 Landmark Sequencing Methods Using Genomes with IncreasedComplexity

This example illustrates an embodiment for providing additional markersin a genome. Additional markers can be useful in genomes that includerepetitive sequences during subsequent assembly steps to generate asequence representation of the genome. Transposon sequences areprepared, each comprising a different barcode. The transposon sequencesare integrated into genomic DNA in a transposition reaction. The genomicDNA comprising the integrated transposon is amplified by whole genomeamplification. A sequencing library is prepared from the amplifiedtemplate nucleic acids. Sequencing data is obtained from the sequencinglibrary. Sequencing reads can include representations of one or morenucleic acids with the same barcode on each nucleic acid. Such nucleicacids are identified as containing sequences that overlap in a sequencerepresentation of the genomic DNA. The sequencing reads can be assembledby identifying barcodes on overlapping sequences.

Example 3 Predicted Average Coverage Using Linked Read SequencingStrategy

Useable fragment lengths are modeled as a truncated exponentialdistribution so that the mean useable fragment length can be obtained bysetting k=b/d, where d is the mean of the non-truncated exponential (thetotal fragment distribution) and b is the value for truncation (either180 or 280 for 100 nucleotides and 150 nucleotides paired end reads,respectively) and then calculating the mean of the truncated exponentialas

E(f)=d(1−(k+1)e ^(−k))/(1−e ^(−k))

The proportion of useable reads is p=C(b)×(1−D(0,T)) where C is theexponential cumulative distribution function, T is the averagerepetitions of observing each fragment (num clusters)/complexity,complexity is the genome size times the number of genome copies dilutedto divided by d, and D is the Poisson cumulative distribution function

Expected length of linked read is then (E(f)−9)×1/(1−p)+9 where p isproportion of useable reads: 9 is subtracted from each read because ofthe reused 9 nucleotides sequence in neighboring fragments; and 9nucleotides is added back to the linked read to account for the endread—one of its 9 nucleotides segments is not shared with another readwithin the linked read.

The distribution of linked read lengths is also exponential with theabove expected value. Very long linked reads can be observed.

Table 1 sets out predicted average coverage for a bacterial genome,human PCR product, and a single copy of a human diploid genome using alinked read strategy.

TABLE 1 Target Average nucleic Genome Read Number of insert Average Meanlinked acid size length Diluted copies clusters distance coverage readlength Bacterial 5.0e6 150 10 35,000,000 50 nt 9.96 X 10,815 nt genomeHuman 20 kb 150 10,000 40,000,000 50 nt 9.96 X 10,811 nt PCR productSingle cell,   3e9 150 2 3000,000,000 50 nt 9.66 X  1,191 nt humandiploid genome

Example 4 De Novo Sequencing Target DNA

This example illustrates an embodiment of assembling sequencing dataobtained from a library of template nucleic acids prepared from a targetDNA.

A plurality of transposon sequences are integrated into the target DNAin a transposition reaction. Each transposon sequence includes a barcodewhich comprises a first barcode sequence and a second barcode sequence.The first barcode sequence is the reverse complement of the secondbarcode sequence. There are more than 10¹⁸ different barcodes in theplurality of transposon sequences, such that the code space is large andout-competes the complexity of the target DNA. Accordingly, eachintegrated barcode is likely to be unique. Optionally, the target DNAcomprising the integrated transposon sequences is sub-sampled. Thesub-sampled target DNA comprising the integrated transposon sequences isamplified by methods of whole genome amplification. A sequencing libraryis prepared from the amplified nucleic acids. Sequencing data isobtained from the sequencing library. The sequencing data comprisessequencing reads for each amplified nucleic acid.

Sequencing reads that include a barcode sequence that can be paired witha barcode sequence of another sequencing read are aligned. Shorteralignments of sequencing reads are aligned with other short alignmentsby identifying paired barcode sequences to produce longer alignments. Asequence representation of the target DNA is generated.

Example 5 Preparing a Linked Library with Blunt-End Barcodes

This example illustrates an embodiment of preparing a linked librarythat includes identical barcodes on template nucleic acids that includeadjacent sequences in a sequence representation of the target nucleicacid.

Transposon sequences are prepared comprising a first fragmentation siteand a second fragmentation site, having a barcode disposed therebetween.Each fragmentation site comprises a site that can be nicked to produce asingle-stranded sticky end, e.g., a restriction endonuclease site thatproduces a single-strand sticky end. The transposon sequences areintegrated into the target DNA by a transposition reaction. Optionally,the target DNA comprising the integrated transposon sequences issub-sampled. The sub-sampled target DNA comprising the integratedtransposon sequences is amplified by methods of whole genomeamplification. The amplified nucleic acids are fragmented at the firstand second fragmentation sites at each integrated transposon sequence togenerate nucleic acids comprising sticky ends. The sticky ends arefilled-in so that each end comprises a barcode. Adaptors are ligated tothe blunt ends of each nucleic acid. The nucleic acids are amplifiedusing primer sites of the adaptors.

A sequencing library is prepared from the amplified nucleic acids.Sequencing data is obtained from the sequencing library. The sequencingdata comprises sequencing reads for each amplified nucleic acid.Sequencing reads that include a barcode sequence that can be paired witha barcode sequence of another sequencing read are aligned. Shorteralignments of sequencing reads are aligned with other short alignmentsby identifying paired barcode sequences to produce longer alignments. Asequence representation of the target DNA is generated.

An exemplary embodiment of this method is depicted in FIGS. 11 and 12.FIG. 11 depicts the insertion of the transposon sequence into the targetnucleic acid (host material), dilution of the template nucleic acids,and subsequent whole genome amplification of the template nucleic acids.The amplified template nucleic acids are fragmented in the presence of arestriction endonuclease that nicks the transposon sequences at thefirst restriction endonuclease site and the second restrictionendonuclease site to yield two sticky ends. FIG. 12 depicts a fill-inreaction of the sticky ends, followed by A-tailing the ends and addingadaptors to the tailed ends. The adaptors can be used in subsequentamplification, library preparation, and methods to obtain sequence data.In other embodiments, the fill-in reaction itself can be used as part ofa detection assay, such as by incorporating detectably labelednucleotides in a variety of assay formats.

The above description discloses several methods and systems of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. For example, the invention has beenexemplified using nucleic acids but can be applied to other polymers aswell. Consequently, it is not intended that this invention be limited tothe specific embodiments disclosed herein, but that it cover allmodifications and alternatives coming within the true scope and spiritof the invention.

All references cited herein including, but not limited to, published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

1. An artificial transposon, comprising: a first transposase recognitionsite, a second transposase recognition site, and a barcode disposedtherebetween, wherein the barcode comprises a first barcode sequence anda second barcode sequence, separated by a linker.
 2. The transposon ofclaim 1, wherein at least one of the transposon recognition sitescomprises a mosaic element.
 3. The transposon of claim 1, wherein thefirst barcode comprises the reverse complement sequence of the secondbarcode.
 4. The transposon of claim 1, wherein the linker comprises afragmentation site.
 5. The transposon of claim 4, wherein thefragmentation site comprises a first nickase recognition sequence and asecond nickase recognition sequence.
 6. The transposon of claim 4,wherein the fragmentation site comprises a restriction endonucleaserecognition sequence.
 7. The transposon of claim 1, wherein the linkercomprises a first primer site.
 8. The transposon of claim 7, wherein thelinker further comprises a second primer sitee and a non-amplifiablesite disposed between the first and second primer sites.
 9. Thetransposon of claim 8, wherein the non-amplifiable site comprises atleast one nucleotide analogue.
 10. An isolated template nucleic acid,comprising at least a portion of a target nucleic acid or copy thereofand at least first and second transposons of claim 1, wherein thebarcode of the first transposon is different from the barcode of thesecond transposon.
 11. The template nucleic acid of claim 10, furthercomprising a first host tag and a second host tag, wherein the targetportion is disposed therebetween.
 12. A method for preparing atransposon, comprising: (a) providing a transposon template nucleic acidsequence comprising a first transposase recognition site, a firstbarcode, and a linker; and (b) extending the transposon templatesequence by hair-pin extension, so that the extended transposon templatesequence further comprises a sequence complementary to the first barcodeand a sequence complementary to the first transposase recognition site.13. A method of preparing a library of transposons, comprisingperforming the method of claim 12, wherein each of the transposons has adifferent barcode.
 14. The method of claim 13, wherein the barcodes arepredetermined.
 15. The method of claim 13, wherein the barcodes arerandomly generated.
 16. A method for preparing a library of templatenucleic acids, comprising: (a) contacting a target nucleic acid with aplurality of transposons of claim 1 under conditions such that a portionof said transposons are inserted into the target nucleic acid.
 17. Amethod for preparing a library of template nucleic acids comprising: (a)contacting a target nucleic acid with a plurality of transposon of claim4 under conditions such that a portion of said of transposons areinserted into the target nucleic acid; and (b) fragmenting said targetnucleic acid at said fragmentation sites.
 18. A method for preparing alibrary of template nucleic acids, comprising: (a) contacting a targetnucleic acid with a plurality of transposons of claim 7 under conditionssuch that a portion of said transposons are inserted into the targetnucleic acid; and (b) hybridizing a primer to the first primer site; and(c) amplifying a portion of the transposon sequence.
 19. A method ofanalyzing a target nucleic acid comprising: (a) performing the method ofclaim 16; (b) obtaining sequence data from said templates; and (c)assembling a representation of a portion of said target nucleic acidfrom said sequence data.
 20. The method of claim 19, wherein assemblingstep (c) comprises identifying the presence of a first barcode sequencein one sequencing read and the presence of a corresponding barcodesequence in another sequencing read, thereby indicating proximitybetween the two sequencing reads in the target nucleic acid.